PRMT5 participates in B cell overactivation in patients with primary Sjogren's syndrome (pSS) through RSAD2‐mediated NF‐κB signaling

Abstract Objective There are new evidences that protein arginine methyltransferase 5 (PRMT5) is widely involved in the progression of various diseases, but its effect is unclear on Primary Sjogren's syndrome (pSS). The main purpose of this study is to explore the regulatory effect of PRMT5 on pSS and its potential mechanisms. Methods CD40L treated CD19 + B cells to construct a cell model of pSS. CCK‐8 assay and Annexin V‐FITC/PI kits were used to measure cell proliferation and apoptosis. ELISA assay was used to determine the contents of IL‐6 and TNF‐α in CD19 + B cells. And commercial kits were used to detect the levels of immunoglobins (IgG, IgM, and IgA) in CD40L‐treated CD19 + B cells. And successfully constructed a pSS mouse model. Results The results revealed an increase in the expression of PRMT5 in CD19 + B cells from patients with pSS. After CD40L treatment, the knockdown of PRMT5 prominently decreased cell viability, the production level of immunoglobulins (IgG, IgM, and IgA), and the content of IL‐10, increased the content of IL‐6 and IL‐8, and promoted the apoptosis of pSS CD19 + B cells. Mechanistically, PRMT5 negatively regulated the RSAD2 and nuclear factor kappa‐B (NF‐κB) signaling pathway. Furthermore, overexpression of RSAD2 and p65 significantly rescued the effect of PRMT5 knockdown on proliferation, immunoglobin production and secreting cytokines in CD40L‐treated CD19 + B cells. More importantly, inhibition of PRMT5 significantly inhibited the symptoms of pSS mice. Conclusions Low‐expression of PRMT5 through inactivation of RSAD2/NF‐κB signalling pathway alleviates the hyperactivity of B cells, which may provide theoretical basis and potential therapeutic targets for clinical treatment of pSS.


| INTRODUCTION
Primary Sjogren's syndrome (pSS) is a systemic autoimmune disease characterized by the involvement of exocrine glands such as salivary and lacrimal glands.It can be divided into pSS and secondary Sjogren's syndrome. 1pSS is a chronic autoimmune disease that is mainly characterized as abnormal activation of B cells. 2 The typical clinical manifestation is xerosis of the mouth and eyes, and it can involve the kidney, lung, nervous system, digestive system, and other systems, resulting in systemic organ involvement. 3Sjogren's syndrome is prevalent in women aged 30−40 years, with an incidence rate of 0.3%−0.4% and 0.77% in the elderly. 4,5rotein arginine methyltransferase 5 (PRMT5), a member of the methyltransferase family, is a type II arginie methyltransferase that catalyzes symmetric dimethylation labeling on histones and non-histones. 6rom gene regulation to human development, PRMT5 is involved in many important biological functions in humans.The role of PRMT5 in various cancers has been fully demonstrated. 7PRMT5 plays a role in promoting cancer in the development of various cancers, including breast cancer, hepatocellular carcinoma, lung cancer, and so forth. 7In addition, PRMT5 can promote the development of inflammation, such as osteoarthritis. 8owever, at present, there is no report on the role and mechanism of PRMT5 in the development of primary dryness, so it needs further exploration.
According to reports, when PRMT5 is inhibited, S-adenosylmethionine domain 2 (RSAD2, a reported antiviral protein) is significantly inhibited.Therefore, PRMT5 may regulate the expression of RSAD2. 9RSAD2, also known as Viperin or Cig5, is a type I interferon mediated virus suppressor protein. 10RSAD2 expression is closely related to immune response and can be used to predict the progression of various autoimmune diseases. 113][14][15] Recent studies have reported that the expression of RSAD2 in CD19 + B cells of patients with pSS is higher than that of non-pSS patients. 16,17However, the role of RSAD2 in the pathogenesis of pSS still needs to be further elucidated.
The main purpose of this study is to explore the role and mechanism of PRMT5 in the occurrence and development of pSS.The results of this study indicate that PRMT5 via RSAD2 mediate nuclear factor kappa-B (NF-κB) signaling pathway inhibits the activity of B cells, improve the symptoms of pSS in mice, and provide preliminary theoretical basis and therapeutic targets for clinical treatment of pSS.

| Blood samples
Forty-seven patients with pSS (pSS group) were selected from the General Hospital of Ningxia Medical University from June 2017 to 2021.All patients were newly diagnosed or did not receive glucocorticoid or immunosuppressive therapy.According to the pSS disease assessment index (ESSDAI), patients with pSS were evaluated. 18There are 27 males and 20 females, aged 35−68 years, with an average age of 44.16 ± 10.35.The course of the disease ranges from 8 months to 16 years.Infection, tumor, renal failure, and other immune diseases were excluded.Forty-seven patients who underwent physical examination in the General Hospital of Ningxia Medical University from June 2017 to 2021 were selected as the control group, from 33 to 69 years old, with an average age of 46.15 ± 12.06 years.There were no autoimmune diseases, no history of other severe diseases, and no family history of autoimmune diseases.There was no significant difference in sex or age between the two groups.This study was authorized by the Ethics Committee of General Hospital of Ningxia Medical University and conducted with the guide of the Declaration of Helsinki.All patients themselves or their families provided written informed consent before sample acquisition (Ethical No. KYLL-2023-0144).

| Construction of an experimental
Sjogren's syndrome (ESS) model mouse C57BL/6 mice aged 8 weeks were purchased from the Experimental Animal Center of Yangzhou University.Mice were raised in a specific pathogen free animal facility, and all experiments were approved by the Living Animal Research and Teaching Committee of General Hospital of Ningxia Medical University.The ESS mouse model was induced according to previously described. 19In short, bilateral salivary glands (SG) were isolated from female mice and homogenized in phosphate buffered saline (PBS) to prepare SG protein.On Days 0 and 7, mice were immunized by subcutaneous injection of SG protein emulsified to a concentration of 2 mg/mL (100 mL per mouse) in equal volume of CFA (Sigma-Aldrich) into the neck.On the 14th day, an enhanced injection was performed at a dose of 1 mg/mL of SG protein emulsified in a Freund's incomplete adjuvant (Sigma-Aldrich).Mice immunized with adjuvant alone served as adjuvant control.Six weeks after immunization we screened ESS model mice based on saliva flow rate (<20 mg/10 min) 20 and selected mice that showed clinical symptoms.All animal experiments were approved by the ethics committee of General Hospital of Ningxia Medical University (Ethical No. KYLL-2023-0502), and were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals.

| Cell proliferation assay
CD19 + B cells were stimulated with CD40L for 72 h, and then were seeded into 96-well plates (1 × 10 4 cells per well) and cultured for 24 h, and 10 µL CCK-8 solution was supplemented for each well.Then, the cells were incubated at 37°C for 2 h, and the absorbance at 450 nm was measured by a SpectraMax M5 fluorimeter.

| Apoptosis assay
After CD40L stimulation, the apoptotic rate was quantitatively analyzed by Annexin V-FITC/PI apoptosis detection kit (BioVision).Treated CDl9 + B cells (5 × 10 5 cells per well) were washed twice by cold PBS and resuspended in 200 μL buffer.Then, adding 10 μL Annexin V-FITC and PI into cell samples, and the samples were incubated at room temperature in the dark for 1 h.At last, apoptotic cells were measured by a flow cytometer (BD Bioscience).

| Enzyme-linked immunosorbent assay (ELISA)
The CD19 + B cells were cultured at 2.5 × 10 5 /mL in RPMI (Miltenyi Biotec) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) upon stimulation with 5 µg/mL soluble recombinant human CD40L (BD Biosciences) in a 24-well culture plate.Centrifuge the cells and collect the cell supernatant.According to the instruction for manufacturers, the concentrations of IgG, IgA, and IgM in the supernatant were measured by using the commercial ELISA kits.The optical density of the samples was read at 450 nm.

| Detection of saliva flow rate
Saliva flow rates were measured as previously described. 21In brief, mice were anesthetized and injected ip with pilocarpine (Sigma-Aldrich) at a dosage of 5 mg/kg body weight.Saliva was then collected using a 20 mL pipet tip from the oral cavity for 15 min.

| Statistical analysis
All experiments had three biological replicates.The data were shown as mean ± SD.All statistics were performed using GraphPad Prism 8.4.2 (GraphPad Software).Tukey's post-multiple tests using ANOVA was selected  to comprise difference of groups.p < .05 was used as a cutoff for statistical difference.

| pSS disease activity index
In the present study, the EULAR ESSDAI score was used to assess the activity of the disease.In the pSS group, the ESSDAI score was higher than that in the healthy group.
Anti-SSA and anti-SSB autoantibody status was assessed in both persons with pSS and controls, and controls were all negative for these autoantibodies.In addition, in the pSS group, 25 persons (53.2%) developed lymphadenopathy, 40 persons (85.1%) suffered from arthralgia, 5 persons (10.6%) with splenomegaly, and 38 persons (80.1%) from cardiopulmonary symptoms.Among people who did not suffer from pSS, 3 (6.4%)demonstrated arthralgia and 2 (4.3%) demonstrated cardiopulmonary arthralgia.The demographic, immunological, and clinical data on the 94 people are presented in Table 1.

| PRMT5 expression was upregulated in CD19 + B cells of pSS patients
The expression of PRMT5 in peripheral blood CD19 + B cells of patients with pSS was detected by qRT-PCR and western blot analysis.The results revealed that the mRNA and protein expression of PRMT5 increased in CD19 + B cells from patients with pSS compared to healthy individuals (Figures 1A,B).We used CD40L to treat CD19 + B cells to construct an in vitro model of pSS.
The results showed that CD40L significantly promoted the cell proliferation of CD19 + B cells in a dose dependent manner (Figure 1C).Compared with the control group, CD40L significantly promoted the protein expression of PRMT5 in a dose dependent manner (Figure 1D).

| Knockdown of PRMT5 inhibits the proliferation of CD19 + B cells and promote apoptosis
The next research investigated the effect of PRMT5 on CD40L-induced apoptosis in CD19 + B cells.As presented in 2 A, si-PRMT5 significantly decreased the protein expression of PRMT5 (Figure 2A).Meanwhile, si-PRMT5 decreased the cell proliferation of CD19 + B cells compared with the si-NC and control group (Figure 2B).In addition, overexpression of PRMT5 significantly increased cell proliferation of CD19 + B cells in cells without CD40L treatment (Figure S1).We also discussed the role of PRMT5 on apoptosis of CD19 + B cells.As presented in Figure 2C level of IL-10 in CD19 + B cells significantly decreased after transfection with si-PRMT5 (Figure 3D).As expected, si-PRMT5 increased the levels of IL-6 and IL-8 compared to si-NC and the control group (Figure 3E,F).
The results showed that knockdown of PRMT5 notably inhibited the cytokines and immunoglobulin production of CD19 + B cells.

| Knockdown of PRMT5 inhibits the expression of RSAD2/NF-κB signal related proteins
In the following study, we will explore the potential mechanism of PRMT5 in the progression of pSS.It has been reported that PRMT5 regulates the expression of RSAD2. 9The results showed that the protein level of RSAD2 was significantly increased in CD19 + B cells from patients with pSS compared to healthy individuals (Figure 4A).In addition, the mRNA expression of RSAD2 was significantly positively correlated with the expression of PRMT5 in the serum of patients with pSS (Figure 4B).Importantly, inhibition of PRMT5 significantly reduced protein expression of RSAD2, p65, and p50 compared to si-NC and control groups in CD19 + B cells (Figure 4C).si-PRMT5 on the content IL-6 and IL-8 in CD40L-induced CD19 + B cells (Figure 5E).

| Inhibition of PRMT5 expression in vivo alleviated Sjogren's syndrome in mice
The model of ESS was established by immune induction of SG protein in mice.Mice were randomly divided into three groups: the first group (control group, n = 10) was immunized with adjuvant alone; the second group was pSS model group: mice were immunized with SG protein; the third group (model+shPRMT5: sh-PRMT5 was transfected with lentiviral vector and induced by SG protein.As presented in 6A, the salivary flow rate decreased significantly in the model group, while sh-PRMT5 significantly promoted the salivary flow rate (Figure 6A).Further data demonstrated that sh-PRMT5 significantly decreased the content of immunoglobin IgG, IgM, and IgA compared with model group (Figure 6B).Interestingly, the results showed that sh-PRMT5 significantly inhibited the content IL-10, and promoted the content IL-6 and IL-8 (Figure 6C).These results indicate that inhibition of PRMT5 in vivo significantly improved the pSS.Meanwhile, sh-PRMT5 significantly inhibited the expression of RSAD2, p65, and p50 in mice (Figure 6D,E).

| DISCUSSION
Our study found that Inhibition of PRMT5 inhibited the activity of B cells induced by CD40L; It can also alleviate the inflammatory response, apoptosis, and immune response of B cells induced by CD40L through RSAD2 mediated NF-κB signal path.In addition, inhibition of PRMT5 in vivo can alleviate the symptoms of Sjogren's disease in mice.It is suggested that PRMT5 may be a potential target for the treatment of pSS.The role of PRMT5 in various diseases has attracted more and more attention in recent years.PRMT5 regulates many cellular processes through its methyltransferase activity. 7,22It specifically catalyzes the methylation of arginine residues in histones and nonhistones. 23More and more evidence showed that PRMT5 may be a oncogene, which can drive the growth and metastasis of cancer cells by epigenetic silencing of some tumor suppressor genes. 24In addition, NF-κB is a ubiquitous inducible transcription factor, the p65 subunit of can be modified by arginine methylation through PRMT5, thus regulating the occurrence of inflammatory response. 25However, there are few studies on the physiological role of PRMT5 in pSS.Our study is the first to confirm the important role of PRMT5 in pSS.In this study, we found a new role of PRMT5 in pSS, and proved that inhibition of PRMT5 expression can inhibit the progress of pSS.Our study found that PRMT5 was upregulated in CD40L-induced CD19 + B cells, and inhibition of PRMT5 alleviated CD40L-induced production of cytokines and apoptosis.Through the above studies, we have reason to believe that PRMT5 may be a key target for the treatment of inflammatory diseases.Although there is no drug therapy directly targeting PRMT5, some inhibitors are expected to be used in the treatment of inflammatory diseases.
][14] In this study, we found that CD40L could upregulate RSAD2 expression in pSS CD19 + B cells.Coincidentally, Zhang et al. found that RSAD2 was increased in CD19 + B cells of patients with systemic autoimmune diseases such as pemphigus and systemic lupus erythematosus [15][16][17] In addition, RSAD2 is highly expressed in CD4 + T cells of patients with rheumatoid arthritis, and its expression is significantly correlated with the clinical characteristics of the disease. 13,26Our previous results showed that RSAD2 regulated the proliferation of CD19 + B cells, the production of immunoglobulin and the expression of IL-10, suggesting that RSAD2 may play a role in pSS by regulating the activity of B cells. 27F-κB signal pathway plays an important role in the biological processes of immune response, inflammatory response, cell proliferation and apoptosis, lymphocyte development, and so on. 28There is growing evidence of NF-κB signal pathway is involved in the pathogenesis of pSS.It has been reported that compared with the healthy control group, the peripheral blood of patients with pSS is lower IκBα.In addition, NF-κB pathway promotes inflammation and induces apoptosis of human salivary gland epithelial cells in pSS. 29In addition, NF-κB pathway is the main regulatory factor of gene transcription, and it is involved in the proliferation, survival, and differentiation of CD19 + B cells.A recent study has shown that activation of p65 phosphorylation can promote the proliferation of peripheral blood B cells and the secretion of immunoglobulin in patients with pSS. 30In this study, NF-κB signal pathway is involved in the regulation of pSS by PRMT5 in vivo and in vitro.

| CONCLUSIONS
Our research shows that PRMT5 inhibition is achieved by inhibiting RSAD2/NF-κB signal pathway to attenuate the overactivity of CD19 + B cells in vitro.In addition, knockdown of PRMT5 regulates production of cytokines of B cells and promotes apoptosis.More importantly, knockout of P significantly improved the symptoms of PSS model mice.The above results suggest that PRMT5 may become a potential therapeutic target for the treatment of pSS.This study provides a preliminary theoretical basis for the application of PRMT5 in the treatment of pSS.Although this is a gratifying result, there are still some shortcomings in the current research.To fully validate the role of PRMT5, we need to explore more in vivo experiments in mice.In addition, the clinical samples selected for this study were small and not representative.Next, we will focus on whether PRMT5 has the same effect in other related cells.At the same time, use to explore more clinical relevance.

F
I G U R E 1 PRMT5 expression was upregulated in CD19 + B cells of pSS patients.(A, B) qRT-PCR and western blot analysis of the relative expression of PRMT5 mRNA and protein in CD19 + B cells of pSS (n = 47) and healthy control group (n = 47).*p < .05versus control.CD19 + B cells were treated with different concentrations of CD40L (1, 2, 5, and 10 µg/mL) for 72 h.(C) The cell proliferation of CD19 + B cells in different concentrations of CD40L.(D) The relative protein expression of PRMT5 of CD19 + B cells in different concentrations of CD40L."*" means compared with control or normal group p < .05,"**" means compared with control group p < .01.GAPDH was used as an invariant internal control for calculating protein-fold changes.PRMT5, protein arginine methyltransferase 5; pSS, primary Sjogren's syndrome.
Knockdown of PRMT5 inhibits the proliferation of CD19 + B cells and promote apoptosis.CD19 + B cells were transfected with control si-RNA or si-PRMT5 for 24 h, and then treated with 5 µg/mL CD40L for 72 h.(A) The relative protein expression of PRMT5 of CD19 + B cells in different group (control, si-NC, si-PRMT5-1, or si-PRMT5-2).(B) The cell proliferation of CD19 + B cells in different group.(C) The apoptosis ratio of CD19 + B cells in different group.(D) The protein expression of caspase-3 in different group.(E) The protein expression of Bax and Bcl-2 in different group.(F) Western blot analysis was used to measure the expression of PRMT5, caspase-3, Bax, and Bcl-2."*" means compared with control group p < .05,and "#" means compared with si-NC group p < .05.GAPDH was used as an invariant internal control for calculating protein-fold changes.PRMT5, protein arginine methyltransferase 5.

3 . 4 |
, inhibition of PRMT5 promoted the apoptosis of CD19 + B cells, as well as increased the protein levels of caspase-3 and Bax, and decreased the Bcl-2 expression (Figure 2C−F).The results indicated that inhibition of PRMT5 could promoted the apoptosis of CD19 + B cells.Knockdown of PRMT5 inhibits the cytokines and immunoglobulin production of CD19 + B cells Next, we investigated the effect of PRMT5 on production of cytokines of CD40L-treated CD19 + B cells.We found that PRMT5 siRNA significantly reduced the levels of immunoglobulins IgG, IgM, and IgA in CD40L-induced CD19 + B cells (Figure 3A−C).At the same time, the F I G U R E 3 Knockdown of PRMT5 inhibits the production of cytokines and immunoglobulin of CD19 + B cells.CD19 + B cells were transfected with control si-RNA or si-PRMT5 for 24 h, and then treated with 5 µg/mL CD40L for 72 h.(A−C) The supernatant IgG, IgM, and IgA levels of CD19 + B cells in different group.(D−F) The supernatant IL-10, IL-6, and IL-8 levels of CD19 + B cells in different group."*" means compared with control group p < .05,and "#" means compared with si-NC group p < .05.GAPDH was used as an invariant internal control for calculating protein-fold changes.PRMT5, protein arginine methyltransferase 5.

3. 6 |
RSAD2-mediated NF-κB pathway participate in the regulation of PRMT5 on CD19 + B cells Further research found that overexpression of RSAD2 or p65 significantly reversed the inhibition of RSAD2 on CD19 + B cells activity (Figure 5A).Meanwhile, pcDNA3.1-RSAD2,or pcDNA3.1-p65significantly decreased the apoptosis of CD19 + B cells, and downregulated the expression of caspase-3 compared with the si-PRMT5 group (Figure 5B,C).As expected, the levels of immunoglobin IgG, IgM, and IgA were notably downregulated by siPRMT5 in CD40L-treated CD19 + B cells, while, overexpression of RSAD2 significantly reversed the effect of PRMT5 (Figure 5D).Meanwhile, pcDNA3.1-RSAD2or pcDNA3.1-p65partly rescued the effect of F I G U R E 4 Knockdown of PRMT5 inhibits the expression of RSAD2/NF-κB signal related proteins.(A) Western blot analysis of the relative expression of PRMT5 mRNA and protein in CD19 + B cells of pSS and healthy control group.(B) Scatter plot showing fitted values and intervals for the PRMT5 mRNA expression in CD19 + B cells and RSAD2 expression in serum from pSS patients.CD19 + B cells were transfected with control si-RNA or si-PRMT5 for 24 h, and then treated with 5 µg/mL CD40L for 72 h.(C) The relative protein expression of RSAD2, p50, p-p65, and p65 of CD19 + B cells in different group (control, si-NC, si-PRMT5-1, or si-PRMT5-2)."*" means compared with control group p < .05,and "#" means compared with model group p < .05.GAPDH was used as an invariant internal control for calculating protein-fold changes.NF-κB, nuclear factor kappa-B; PRMT5, protein arginine methyltransferase 5; pSS, primary Sjogren's syndrome.

F
I G U R E 5 RSAD2-mediated NF-κB pathway is involved in the regulation of si-PRMT5 on CD19 + B cells.CD19 + B cells were transfected with si-PRMT5 or pcDNA3.1-RSAD2or pcDNA3.1-p65,and then treated with CD40L (5 μg/mL) for 72 h.(A) The cell proliferation of CD19 + B cells in different group.(B) The apoptosis ratio of CD19 + B cells in different group.(C) The protein expression of caspase-3 in different group.(D) The supernatant IgG, IgM, and IgA levels of CD19 + B cells in different group.(E) The supernatant IL-6, IL-8, and IL-10 levels of CD19 + B cells in different group."*" means compared with control group p < .05,and "#" means compared with si-PRMT5 group p < .05.GAPDH was used as an invariant internal control for calculating protein-fold changes.NF-κB, nuclear factor kappa-B; PRMT5, protein arginine methyltransferase 5.

F I G U R E 6
Inhibition of PRMT5 expression in vivo alleviated Sjogren's syndrome in mice.The model of experimental Sjogren's syndrome (ESS) was established by immune induction of salivary gland (SG) protein in mice.Mice were randomly divided into three groups: the first group (control group, n = 10) mice were induced with adjuvant alone; the second group (pSS model group, n = 10) mice were induced with SG proteins; the third group (model + shPRMT5; n = 10) mice were induced with SG proteins and transfected with lentiviral vector sh-PRMT5.(A) Salivary flow rate in control, model, model+shPRMT5 group.(B) The levels IgG, IgM, and IgA in serum of mice in each group.(C) The levels IL-10, IL-6, and IL-8 in serum of mice in each group.(D) The protein expression of PRMT5 and RSAD2 in control, model, model+shPRMT5 group.(E) The protein expression of p50, p-p65, and p65 in control, model, model+shPRMT5 group."*" means compared with control group p < .05,and "#" means compared with model group p < .05.GAPDH was used as an invariant internal control for calculating protein-fold changes.PRMT5, protein arginine methyltransferase 5; pSS, primary Sjogren's syndrome.